M AY 2 0 1 6

Advancing
GC Methods and
Environmental
Analysis

IntroDUCTION

as chromatography (GC) is a powerful technique for environmental

analysis. The articles in this new e-book look at both the technique and
its application, providing essential information about GC fundamentals
as well as an example of how the technique can be used in new ways in
environmental analysis.
First, Raquel Fernndez Varela talks about her use of gas chromatographymass
spectrometry (GCMS) for the analysis of marine pollution. She used GCMS to study
how marine polychaetes respond to contamination from oil spills. Her method had to
address important challenges related to the wide range of sample compounds involved
as well as complex data analysis.
Next, John Hinshaw presents a GC Troubleshooters Toolkit. This guide to the
essential tools for installing, maintaining, and troubleshooting GC instruments and
columns not only explains the proper use of traditional laboratory tools, but also offers
some surprising suggestions for using unconventional items in the GC lab, including
some you never think of as tools at all.
Our last piece provides practical information about how to choose the right GC column
for a given analysis. The article provides five overall selection rules, as well as advice
about choosing physical column dimensions such as length, internal diameter, and film
thickness. It also reviews the key analyte and stationary-phase interactions that must be
considered.
We hope you enjoy this e-book, and find it helpful in your work in
environmental analysis.

TOC

Table of contents

Advancing GC Methods AND

Environmental Analysis

Marine Pollution Analysis

Untargeted Metabolomics
for Marine Pollution
Analysis Using GCMS

04

An interview with Raquel Fernndez Varela

Troubleshooting

A GC Troubleshooters Toolkit
John V. Hinshaw

09

GC Column Selection

Pragmatic Rules for

GC Column Selection

20

Tony Taylor

Untargeted Metabolomics
for Marine Pollution
Analysis Using GCMS

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4 | May 2016 | LCGC

Marine polychaetes are a common type

of annelid worm widely spread in marine
environments. In this interview, Raquel
Fernndez of the University of Copenhagen,
Denmark, talks about her innovative approach
to developing an untargeted method to monitor
polychaetes and to assess their potential use in
environmental monitoring of oil spills.
You recently developed a gas chromatography
mass spectrometry (GCMS) metabolomics
platform to investigate marine polychaetes.
Why are you investigating marine polychaetes?
The idea arose after my PhD, which dealt with
monitoring and characterizing the oil spill that
resulted from the 2002 Prestige oil tanker
disaster off the coast of Galicia, in northwest
Spain. I then applied for an IEF Marie Curie
Fellowship with Prof. J.H. Christensen at the
University of Copenhagen (Copenhagen,
Denmark).
One of the requirements of the fellowship
was that that the project had to be innovative,
and despite the fact that marine polychaetes
are ubiquitous in the marine environment, no
studies had been performed to investigate
the potential of these organisms for biological

EyeEm/Getty Images

Quick Guide to
Consumables for the
TRACE 1300 GC

Marine Pollution
Analysis

Troubleshooting

monitoring. My objective was to examine

how hydrocarbon pollution and other
environmental stressors control the
metabolic response at a molecular
level and evaluate their use as pollution
indicators. If a response could be
determined, the limited mobility and
tolerance to polluted conditions of
marine polychaetes would make them
an excellent candidate for biological
monitoring.
What were the limitations of existing
analytical tools when developing this
method?
The main limitation was that there was
no analytical platform that could cover
all the compounds. This was, and still is,
especially true for untargeted studies
in which the interest can span multiple
classes of metabolites. When the
study began we looked at the existing
analytical platforms and after an initial
screening, we first thought that ultrahighperformance liquid chromatography
mass spectrometry (UHPLCMS) would
be the best candidate for our purposes.
However, the data analysis and the
preprocessing turned out to be especially
problematic because the shift pattern
in retention time and sheer number of
samples proved to be too complex a
problem to be adequately corrected
using the bioinformatics tools we had at
the time. Hence, we decided to move
from liquid chromatography (LC) to gas
chromatography (GC), because GC tends
to be more stable in terms of retention

5 | May 2016 | LCGC

GC Column
Selection

time. This implied that the separation

criteria and targeted class of compounds
of primary interest also shifted from
more watersoluble to more hydrophobic
compounds. The unsuitability of GC
for nonvolatile or thermally unstable
compounds also required a suitable
derivatization scheme before we could
proceed with the GC analysis.
What is novel about your approach?
And what challenges did you have
to overcome from an analytical
perspective?
The most novel aspect is that we
presented an entire method for
environmental metabolomics in marine
worms from sample preparation and
extraction, to chemical analysis, data
preprocessing, and multivariate methods.
We included all the steps needed to
perform a good untargeted analysis.
One, if not the biggest, challenge we
had to face was the optimization of
the parameters for the preprocessing
using XCMS. XCMS is free open-source
software to process metabolomics data
that includes feature detection, retention
time alignment, and feature matching
across samples to generate a consensus
table of features common to a majority
of the samples. The package is mature
and it has been successfully used in many
metabolomics studies. This software suite
was developed initially to preprocess LC
MS-based data, and to our knowledge,
it is used predominantly for this purpose.
However, with some optimization to the

Marine Pollution
Analysis

Troubleshooting

parameters, it can be successfully used

for treating GCMS-based metabolomics
data, as we show in our approach.
We worked for days on testing the
different algorithms available in XCMS
for feature detection, peak matching,
and retention time alignment; different
combinations of parameters; and
even developed in-house a number of
additional software tools and diagnostics
to check the results but yet, we could
not find a combination that worked for
all the samples at once and satisfied
us entirely. In fact, the difficulties in
preprocessing were the main reason why
we shifted to GC in the first place.
What were your main findings?
The main finding was that a specific
metabolite pattern variation in marine
polychaetes could be correlated with
oil exposure, meaning that metabolite
patterns could serve as oil pollution
indicators in the future. However, no
biological variation was taken into account
in this study and only the remaining data
we collected during the project could
throw some light on this. Another result
that I really think is important, though
it might not appear as such at first
glance, is that we really understood the
importance of the incorporation of quality
control (QC) samples (for example, a
pooled sample) not only for the chemical
analysis, but also for the quality of the
preprocessing, to reduce analytical
variation and to quantitatively determine
analytical precision. Finally, we showed

6 | May 2016 | LCGC

GC Column
Selection

how the normalization of the analytical

profiles is essential to the proper profiling
of metabolic data; we compared three
different approaches to investigate which
one is the most suitable for our particular
case.
Are there any specific problems
encountered when developing an
untargeted GCMS method?
The main problem in untargeted
GCMS metabolomics is achieving a
wellresolved chromatographic separation
of a large number of metabolite species
with different chemical and physical
properties in a single analytical run before
performing the data preprocessing. Also,
the very heterogeneity of the data made
the optimization and validation of the
parameters for the data preprocessing
especially difficult, yet essential to the
whole method. And, again, it was only by
using the QC samples that we achieved
our results. The comparison of the XCMS
results against manual integration of
selected peaks relative to different classes
of compounds showed an excellent
correlation. It is evident that XCMS
offers optimal choice of parameters to
provide an accurate feature extraction,
comparable with manual peak integration.
Is there any general advice you would
offer to chromatographers attempting to
develop untargeted methods?
One of the biggest issues in
metabolomics is reproducibility. Often
the technical variation is high, despite

Marine Pollution
Analysis

Troubleshooting

the fact that no biological variation could

be observed in this study. We managed
to show the need to include a large
number of QC samples in each batch
or sequence. Even though many have
pointed this out, I believe this cannot be
stressed enough! Another important point
is not to underestimate the power and
the effect of proper data preprocessing.
Also, in general, it can be extremely time
consuming. For space reasons, it may not
take too much space in published works,
and it often looks simpler and easier than
it actually is. It is not pointand-click! It
requires knowledge about your samples,
rightthinking choices, and awareness of
the consequences it may have on the data
analysis. In this respect, I think, the third
and most important point is this: Always
go back to the original data and doublecheck that what you get from the data
analysis shows in the raw data.
Are you developing this project further?
I believe that the method we developed
can be used in real-life studies. This
study was limited by the experimental
design, which did not allow us to
distinguish biological variation. We have
additional data where this aspect was
taken into account; these data seem
to show that we could separate marine
polychaetes exposed to different levels
of contamination. The data analysis
though is not complete and we need to
thoroughly validate the results.

7 | May 2016 | LCGC

GC Column
Selection

The plan is to write them into one or

two additional papers, but my current
involvement in other projects in microbial
metabolomics at Chr Hansen A/S (Denmark)
makes it difficult to do it right now.
Raquel Fernndez Varela has a M.Sc. and
a Ph.D. degree in analytical chemistry, both
from University of A Corua (Spain). She
moved to Denmark in 2011 with a Marie
Curie Intra European
Fellowship to performed
postdoctoral studies
at the Department of
Plant and Environmental
Sciences at the University
of Copenhagen
(Denmark). Her maint
research at that point was to examine
how pollution and other environmental
stressors control metabolite response
in selected organisms at the molecular
level. In September 2014 she moved to
Chr Hansen A/S (Denmark), where she is
currently working as a research scientist in
microbial metabolomics. She has extensive
experience in gas chromatography,
mass spectrometry, applied multivariate
methods, and environmental and microbial
metabolomics.
How to cite this article: Untargeted Metabolomics
for Marine Pollution Analysis Using GCMS, The Column,
September 8, 2015, pp. 25.

Protecting the future while

saving bottom lines. Its a

BALANCING
act for a brand new age
.

It happens every day in labs all around the world.

Theres the need to meet ever-evolving compliance standards and regulations in environmental
analysis determinations. Balanced with the need to do those determinations in a world of
tightening budgets and cost containment.
But what if there was a way to satisfy both?

Everything designed to handle your toughest determinations from trace to complex matrices not

Protecting the future.

A GC Troubleshooters
Toolkit
John V. Hinshaw

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9 | May 2016 | LCGC

Here, we present tools and accessories that gas

chromatographers use in the laboratory.
Some years have passed since this list was last
published in LCGC (1). During the interim, my
toolbox has gained some new items, and a few
others have seen little or no use. Given the modern
obsession with list-making, readers may enjoy this
update and perhaps learn about some tools they
might like to add or replace in their own toolkits.
Every profession has its specialized tools.
Those used in chromatography often are just
as specialized as those used in computer
repair or automotive work. Many of the tools
and accessories that gas chromatographers
keep on hand for installing, maintaining, and
repairing their chromatographs are also found
in the toolkits of plumbers, carpenters, and
homeowners. Wrenches, screwdrivers, pliers,
and metal-tubing cutters are some easily
recognized examples. Other items such as dental
instruments or paper correction fluid are familiar,
but their use in the laboratory environment
might not be immediately obvious. Still others,
like a column flowmeter, septum nut wrench, or
a specialized fused-silica column cutter, arent
found outside the laboratory at all.

Andy Sack/Getty Images

Gas Chromatography
Liner Selection Guide

Marine Pollution
Analysis

Troubleshooting

Here, then, is the list of todays

tools and accessories along with
some information about their use and
significance. One or more specialty
manufacturers offer many of these
chromatography-specific items in their
catalogs or on-line offerings. I scanned
through several catalogs and web sites
and gleaned some new items that I have
included here.

Butane Lighter

A butane lighter is a convenient source

of hydrocarbon gas for measuring an
approximate unretained peak time.
Butane is effectively unretained at
temperatures above 75 C on liquidphase coated columns with phase ratios
above 50. Columns at low temperatures
or with lower phase ratios (thick stationary
films) may retain butane and separate the
traces of ethane and propane present
in the butane fuel. Use the earliest
observable peak for the best estimate
of unretained peak time. Natural gas is
mostly methane; if your laboratory has
a supply of natural gas (mine doesnt)
it makes a good substitute for a lighter
and is less retained than butane. Just
be sure to turn off the gas after youve
filled a syringe with it. A lecture bottle
of methane with a suitable pressure
regulator is another excellent source of
the unretained substance. Concentrations
in the low percent range work well.
Hydrocarbons wont work for unretained
peak time measurements with electroncapture detection (ECD). Instead, try

10 | May 2016 | LCGC

GC Column
Selection

loading the syringe with a puff from a

pressurized can of dust remover such
as Dust-Off, which contains 100%
1,1-difluoroethane.
Hydrogen or heliumwhichever is not
the same as the carrier gasmake good
unretained peak markers for porous
polymer or molecular sieve columns that
retain hydrocarbons strongly, plus these
two gases should be readily available
in most gas chromatography (GC)
laboratories. Flame ionization detection
(FID) will not respond to hydrogen or
helium, but other detection methods
such as thermal conductivity detection
(TCD), pulsed discharge detection (PDD),
or helium ionization detection (HID), the
latter with hydrogen as the unretained
substance, should respond well.

Cable, Three-Wire

Recorder cable finally has gone the way

of chart recorders: You might find some
lying around but they havent been used
in years. I have one or two in the lab just
in case the need arises. I keep some
spare USB cables as well.

Correction Fluid

Use white correction fluid to mark the

measured position on a column that
corresponds to the correct column
penetration depth into an inlet or
detector. Measure the depth after
inserting the column into the nut and
ferrule and making a fresh cut on the
column end. Some regulated laboratories
policies dont permit the use of correction

Marine Pollution
Analysis

Troubleshooting

fluid for data integrity reasons. A septum

into which a slot has been cut may be
slid onto the column below the nut and
ferrule to act as a positioning aid. Remove
the septum before heating the column
oven. A positioning gauge (see Ruler
below) is a good alternative. I also have
used a black permanent marker for this
purpose; just be careful not to get it on
your fingers or clothing.

Cutters, Fused-Silica Column

The best fused-silica column cutting tool

is the one that holds the column in an
adjustable chuck and cuts with a diamond
chip as the operator rotates a thumb
wheel. This tool also has a magnifying
glass on the opposite end for inspecting
the fresh cut for squareness and lack
of burrs or hanging polyimide coating.
A pen-like tool with a sapphire tip or
ceramic scoring wafers or scribes that
make a sharp cut on the column so that it
may be broken cleanly in two are the best
inexpensive alternatives. In any case, a
fresh cut should be made and inspected
just before placing the column into the
inlet or detector and after sliding on the
nut and ferrule.

Cutters, Tubing

A large plumbers tubing cutter for 1/4in. metal tubing and a smaller one for
1/8-in. tubing are used extensively during
instrument installation and gas-supply
setup. The correct 1/8-in. size cutters are
available from some chromatography
supply companies dont try to use the

11 | May 2016 | LCGC

GC Column
Selection

mini cutters found in home supply stores.

Keep a supply of new cutter blades on
hand. These disks wear out rapidly, and a
dull blade will distort the tubing diameter
and make it difficult to slide on the back
and front ferrules. Power cutters with
high-speed rotary abrasive cutting wheels
are also available. These are mandatory
for cutting 1/16-in. tubing for low-deadvolume gas valving applications, and
they work well for any tubing up to 1/4in. diameter. After cutting and squaring
the tubing (see Tubing Reamer and
Deburring Tools below) be sure to
clean out the ends with a solvent such
as isopropanol so that loose particles
cant get into the instrument components
or interfere with the passage of peaks
through column ends.

Deactivated Fused-Silica Tubing

Gas chromatographers can use 5 or 10 m

of deactivated 0.53-mm i.d. fused-silica
tubing with a press-fit connector as a
retention gap when necessary. Shorter
pieces can act as a column-to-detector
adapter when you dont want to put the
coated column end into a detector to
avoid column bleed from the column end
at hot internal detector temperatures.

Dental Mirror

A plastic dental mirror with a frontsilvered surface makes it easy to examine

the underside of an inlet fitting in the
oven, or to check other inaccessible areas
for loose or missing parts. The mirror
can also be used to detect the flame in

Marine Pollution
Analysis

Troubleshooting

a flame ionization or flame photometric

detector by observing condensation
of emitted water vapor on the cool
mirror surface. A shiny wrench is a good
substitute for the mirror in this case.

Dental Pick

A dental pick is very handy for removing

septa from septum nuts and debris such
as bits of graphite ferrule from fittings.

Diagonal Cutters

Diagonal cutters are used only for cutting

electrical wires. They should not be used
as a substitute for a tubing cutter. Dont
even think about threatening your fusedsilica column with one!

Eyedropper, Plastic

I have a box of these that is in frequent

demand. I use them to place small drops
of isopropanol onto fittings for leak
checks. Some types have rough volume
indications more like a pipette, and I have
used them to make a crude dilution of
qualitative test mixtures when accuracy
wasnt required.

Ferrules, Capillary Column

A good assortment of graphite and

graphiteVespel capillary column ferrules
is essential. Keep at least 10 of each inner
diameter in the fitting sizes that match
your instruments oven fittings. In a pinch,
graphite ferrules can be squeezed into
sealing on smaller columns than they
were designed for. Its always a good idea
to install new ferrules with a new column;

12 | May 2016 | LCGC

GC Column
Selection

old ferrules can be reused on the same

column if a seal can be made without
over-tightening the fitting.

Ferrules, Metal Tubing

Ferrules for metal tubing are also essential. I

used to prefer brass ferrules for copper and
stainless-steel tubing, but nowadays I match
the ferrule material to the tubing because
some brass ferrules will bind to stainless
steel upon tightening. Some instruments
use 1/16-in. or 3/32-in. stainless tubing. This
tubing is best connected using 1/8-in.
graphiteVespel reducing ferrules for 1/8-in.
fittings, or 1/16-in. ferrules for the 1/16-in.
tubing. Chromatographers should be aware
of the potential for atmospheric oxygen
contamination of the carrier gas from
improperly installed supply tubing and
ferrules. Even with the best filtration in
place, a leak between the filters and the
instrument will nullify the effect of the filters.

Files, Needle

An assortment of needle files can be used

to pick out ferrules from fittings, remove
burrs, and shape the ends of metal tubing
before it is connected to a fitting. Dont
forget to clean off all traces of metal
before connecting.

Flexible Magnetic Pickup

A flexible 2-ft magnetic pickup comes in

handy when you drop a small part inside
the instrument. Another similar tool has
a three-jawed claw operated by a
plunger, and it will pick up nonmagnetic
items.

Marine Pollution
Analysis

Troubleshooting

Flowmeter, Electronic

An electronic flowmeter is an expensive

investment, but I believe that it will pay
for itself many times over with improved
accuracy and precision over bargain-priced
bubble flowmeters. I prefer the type of
electronic meter that senses flow directly
and that allows the operator to select the
type of gas in use, such as air, helium, or
hydrogen. The option to calculate split
ratios from the measured split vent and
column flows is a handy feature.

Flowmeters, Bubble

If you use bubble flowmeters, keep two

sizes on hand. The large size is good
for measuring FID air or inlet split vent
flows up to several hundred milliliters
per minute. The smaller size is better for
packed-column or hydrogen flame-gas
flows in the 1050 mL/min range. Dont
try to use a bubble flowmeter to measure
capillary column flows below 10 mL/min.
The carrier gas will diffuse out of the
bubble and you will get a low reading.
Measure the unretained peak time
instead and calculate the flow rate from
it. Note that this calculated flow rate or
the rate displayed by electronic pressure
control will only be as accurate as the
column dimensions the operator uses.
See an earlier GC Connections (2) for a
detailed discussion.

Glass Wool Insertion

and Removal Tool

This item is useful for those who must

install glass wool in inlet liners, or for the

13 | May 2016 | LCGC

GC Column
Selection

hardy few who pack their own columns

and use glass wool to hold in the packing.
These days I find little use for it.

Inlet Liners

Inlet liners are often broken or chipped

during installation or removal. They also
can become contaminated with sample
residue or may lose their deactivation if
used for too long at high temperatures.
Keep some spares on hand, both for
packed inlets and for split or splitless
injections. If you use deactivated liners,
it is better to purchase them already
deactivated than try to treat them yourself
because of the chemical hazards and
waste disposal problems this will create.

Inlet Liner Removal Tool

A tapered high-temperature silicone

rubber tool on a metal holder does a
good job of grabbing glass inlet liners
and removing them without cracking
or chipping the liner top. Most GC
instrument manufacturers will supply
specific tools and instructions for a
particular inlet option.

Leak Detector, Electronic

An electronic leak detector is expensive,

but it is indispensable for finding small
leaks around hot fittings or inlets on
which a liquid cannot be used (see LeakChecking Solution below). The most
sensitive type of leak detector uses a small
pump to pull air from a probe through a
thermal-conductivity cell. The presence
of carrier gas or hydrogen changes the

Marine Pollution
Analysis

Troubleshooting

thermal conductivity and causes a change

in the detectors readout compared to a
reference air flow. Sensitivity for nitrogen
carrier is limited. I also have a small
handheld, battery-powered leak detector
that has a series of light-emitting diodes
which indicate the detected leak rate.
This detector is great to carry around
in a laboratory like mine with lots of
instruments and little clear bench space.

Leak-Checking Solution

In my toolbox, the only acceptable leakchecking solution is a small bottle of

pure isopropanol with an eyedropper.
Other solutions may contain material
that can leak into the gas-supply lines or
columns and cause ghost peaks or other
contamination.

Magnifier

A small magnifier is used to examine

freshly-made column or tubing cuts for
burrs or uneven edges.

Manufacturer-Specific Tools

For each GC system, there are always

some specialized tools. These are
used, for example, to open up a split
splitless inlet or to remove the inlet liner.
Perhaps a special wrench is required
for FID system flame jet replacement.
Whatever the case, keep all such tools
with their instrument: you will need them
eventually. Some of the chromatography
suppliers offer their own versions of
these tools, which often are more useful
than the freebie ones that come with the

14 | May 2016 | LCGC

GC Column
Selection

instruments. Several companies have

toolkits for a specific popular instrument
that is a must-have item for me.

Mini Flashlight

A flashlight is very handy for inspecting

the interior of inlets and detectors for
obstructions, as well as for illuminating
the oven interior. I prefer the type with
the bulb on a flexible gooseneck. No one
yet has built a GC oven with a light that
comes on when the door is opened.

Nuts, Capillary Column

There are several different styles of

capillary column nuts that are used in
the GC oven. My toolkit includes some
of each type for my instruments. I never
try to substitute one for another, even
though the thread sizes are the same.

Nuts, Metal Tubing

Nuts for metal tubing are more standardized

than capillary column nuts. Swaged
fittings normally are used for outside
tubing connections and often for internal
connections as well. Keep an assortment of
1/4-in., 1/8-in., and 1/16-in. sizes on hand.
Dont try to mix nuts and fittings from
different manufacturers. Ive picked one
type and tried to purge all the others from
my lab, so I dont have to peer at the small
letters on each fitting to discern its type.

Nut Drivers

Handheld nut drivers are a useful

addition, but I find that I use them more
at home than in the laboratory.

Marine Pollution
Analysis

Troubleshooting

Paintbrush

An artists paintbrush with handle is handy

to clean out debris from small areas inside
detectors or inlets. It can also apply leakchecking solution to fittings, although I
dont recommend this practice because of
potential contamination of the gas stream
with the leak-checking solution.

Paper Clips

Jumbo-size paper clips with smooth

sides are convenient for blocking off
inlet or detector fittings for testing
purposes. Unbend the clip and attach
it to the fitting with a nut and 1-mm i.d.
graphite-vespel ferrule. With the column
connection blocked off, you can pressurecheck an inlet. A detector check can
be run in this manner without column
influences on noise or stability.

Pin Vise and Drills

A small pin vise and a set of drills can be

used in an emergency to drill out a used
ferrule or to enlarge one that is too small
to fit a column. Sometimes the small
drills can help to remove a ferrule that is
stuck in a fitting or to remove debris from
inside fittings or tubing ends.

Pliers

I keep some small needle-nose pliers,

a pair of larger multigrip pliers, and
one pair of locking pliers in my toolkit.
The larger gripping pliers are useful for
holding a straight length of 1/8- or 1/4-in.
metal tubing while cutting it, although I
take care not to grip the tubing anywhere

15 | May 2016 | LCGC

GC Column
Selection

near a location where a connection is to

be made because the scratches from the
pliers would make it impossible to get a
good seal.

Press-Fit Connectors

Glass press-fit connectors make it easy to

repair a broken column temporarily (until
a replacement can be installed). These are
available in many sizes to connect fusedsilica tubing of the same or different
diameter. They also connect a column
with a retention gap. One manufacturer
now offers a vacuummelting device that
makes near-perfect connections.

Pressure Gauge, Inlet

I have a conventional 060 psig pressure

gauge with a syringe needle attached
that I can insert into an inlet through the
septum. Once in a while I need to check
the inlet pressure this way, instead of
relying on the instruments gauges or
electronic pressure readouts.

PTFE Tape

PTFE tape is used sparingly on tanks and

interconnecting fittings where threads
form the seal. Use two layers of tape, not
more, and wrap them around the threads
in the direction the nut tightens so that the
tape will be drawn into the fitting instead
of pushed out. PTFE tape should never be
used in swage-type ferrule-sealed fittings,
where it will only cause a leak, nor is it
used at the high-pressure supply cylinder
connection. Several types of this tape are
available; be sure to select the right one.

Marine Pollution
Analysis

Troubleshooting

Ruler

A small metal ruler measures the correct

column penetration depth into an inlet or
detector. Dont use a plastic ruler, because
it might melt in contact with heated inlets or
detectors. For convenience, make marks on
the ruler that correspond to the correct inlet
and detector depths. Several manufacturers
offer capillary column installation gauges
with the appropriate markings.

Scissors

A good sharp pair of scissors comes in

handy for opening packages of ferrules,
or for making paper stars out of waste
paper thats waiting to be recycled while
watching for peaks to be eluted. Scissors
are not to be used to cut fused-silica
columns (but you can believe that Ive
seen someone try it).

Screwdrivers, Phillips-Head

I found three Phillips-head screwdrivers

in my toolkit: large, medium, and small.
The small one is part of a set of jewelers
screwdrivers with rotating handles.

Screwdrivers, Slotted-Head

I also keep three slotted-head

screwdrivers. The small one is useful
for securing electrical connections to
screw-type terminals. My set of jewelers
screwdrivers in a small plastic box have a
knurled body and a separately-rotating
knob that make it easy to turn the shaft
with one hand. They havent seen too
much GC use, but they are good for
tightening the frames of my eyeglasses.

16 | May 2016 | LCGC

GC Column
Selection

Seals, Inlet

Many capillary inlets use an internal

O-ring seal to isolate the incoming
and exiting split flows. These seals
are available in a variety of materials,
including silicone, graphite, and hightemperature polymer. Worn seals will
cause internal leakage and performance
losses. Keep a good assortment on hand
for each instrument. Some instruments
use a metal seal and washer at the
bottom of splitsplitless inlets. For these,
I prefer the deactivated seals available
from at least one supplier. A seal with a
Vespel seating surface for the inlet liner
was recently made available, as well.

Septa

Spare septa are a requirement. Septa

should be changed often. If you wait
until retention times begin to drift
upward then its too latea significant
leak that will compromise results already
has developed. Keep both normaltemperature range septa as well as some
high-temperature ones on hand. I handle
my septaand all internal inlet parts
with tweezers or cotton gloves: a little
bit of finger contamination can create a
significant baseline bleed level.

Static Pad

A static pad is a grounded, conductive

plastic sheet onto which it is safe to place
electronic components that must be
protected from damaging electrostatic
discharge. Any circuit boards removed
from an instrument should be placed

Marine Pollution
Analysis

Troubleshooting

on a grounded static pad, or in a staticproof bag.

Static Wrist Strap

A grounded static wrist strap prevents

the technician from imparting a
potentially harmful static discharge
into instrumentation or components.
Always wear one when working inside
an instrument or removing components,
and in all cases be quite sure that the
instrument power has been removed
while the instrument itself remains
grounded.

Stopwatch, Digital

A digital stopwatch times bubbles in

a bubble flowmeter, and also times
an unretained peak. Its often more
convenient to use a stopwatch when
setting up an instrument than to operate
the chromatography data system for
each test injection. Select a stopwatch
with readout to 0.01 s. Some GC systems
include a stopwatch function on the
display that includes flow, split ratio, and
linear velocity calculations. These days I
just use my smart phones stopwatch and
timer functions, and then its calculator to
find flow rates or average linear velocities.
Good phone applications are available
with additional chromatography functions.

Syringe

I keep two syringes for setup purposes.

One 10-L syringe is for injecting
methane or butane to measure the
unretained peak time and ascertain that

17 | May 2016 | LCGC

GC Column
Selection

the flame is lit and carrier gas is flowing.

The other is for making liquid test-mixture
injections as part of a column check-out.
Sample syringes are kept separately.

Syringe-Cleaning Wires

Syringe cleaning wires may be used in

an emergency to clear septum particles
or other debris from syringe needles.
I recommend discarding stubborn
contaminated syringes; take steps to keep
the syringe clean instead.

Test Mixtures

Column and detector test mixtures

verify column performance and detector
sensitivity. Keep a fresh vial of each
type on hand. Column test mixtures are
available for polar and nonpolar capillary
columns, and there are test mixtures for
each detector type. Some manufacturers
provide a detector test mix that combines
components for testing several different
detectors. After they have been opened,
test mixtures can be kept for a while
in septum-sealed vials. Their lifetime is
limited because of gradual evaporation. If
you keep test mix in a vial, remove the vial
cap rather than puncturing the septum
when withdrawing liquid for injection.
Some laboratories find it more convenient
to keep dilute test mixtures on hand
because these are more easily disposed
of than the concentrated mixes. Many
laboratories have their own qualification
and validation standards, of course, but
the manufacturers mixtures allow easy
comparison to the factory test results.

Marine Pollution
Analysis

Troubleshooting

Tube Bender

I use this simple tool to make controlled

bends of copper or stainless steel
tubing for connecting the supply tanks
to the filters and then to the back of the
instrument. Tube benders come in sizes
to fit standard tubing diameters.

Tube Reaming and

Deburring Tools

These tools are used to remove burrs

and irregularities from metal tubing after
cutting. They are available for the standard
tubing diameters, and I highly recommend
using them to ensure leak-free connections.

Tubing, Plastic and Rubber

I keep several pieces of black and clear

silicone rubber tubing on hand for
connecting my flowmeter to column
ends, split vents, and other flow sources.
The narrower pieces of tubing fit inside
the wider ones so that I can adapt
the flowmeter fitting to a variety of
connections. Of course, I never use plastic
or rubber tubing for any gas at elevated
pressure or for permanent supply or
internal connections.

Tweezers and Hemostats

A pair of tweezers can hold small nuts

or ferrules without risking contamination
with skin oils or a burn from hot items.
Some tweezers have a convenient locking
feature that frees one hand for other
tasks, as will a spring-loaded hemostat.
Rubber tips help hold fragile capillary
columns or inlet liners.

18 | May 2016 | LCGC

GC Column
Selection

Vial Crimper

Vial crimpers attach aluminum crimp-top

seals to autosampler vials. Several crimptop sizes are commonly used for GC: 8
mm for 0.8-mL vials, 11 mm for 1.5- or
2.0-mL vials, and 20 mm for 5-mL and
20-mL vials. Hand crimpers are the least
expensive, and some are available with
interchangeable jaws that accommodate
different vial sizes. Automated benchtop
crimpers are less mobile, but the jaws
can be interchanged quickly and they are
best for laboratories with high sample
throughput.

Vial Decapper

Vial decappers perform the opposite

function of a crimper: They remove crimptop seals from vials. Decappers come
in the same sizes as the crimpers and
resemble a pair of pliers. Some caution
is required when using these so as not
to break the neck of the vial. After the
caps are removed, the contents may be
properly disposed. Some laboratories
reuse sample vials, but I recommend a
fresh vial for each sample if at all possible.

Vial, Autosampler

I keep a spare autosampler vial to check

for carrier gas flow during column
installation. Fill the vial halfway with
distilled water and then insert the column
outlet after connecting to the inlet and
turning on carrier gas pressure. The
presence of bubbles shows positive
carrier gas flow.

Marine Pollution
Analysis

Troubleshooting

GC Column
Selection

Wipes, Laboratory

Wrenches, Adjustable

Wire Brushes

Conclusion

I wet these with some isopropanol and

then clean any debris or oil off the ends
of capillary columns before inserting them
into inlets or detectors. They also are
handy for tipping a drop of test mix off a
syringe needle, if disposed of properly.
Paper towels dont work as well: They
may leave fibers behind, and they could
deposit a chemical residue.

Wire brushes can dislodge particles and

debris from detector parts and some
sealing surfaces. Be careful not to score
polished metal surfaces, or damage
ceramics. It is better to replace a severely
dirty FID flame jet or collector than to
clean it forcibly.

Wrenches, Hexagonal and Star

A full set of inch and metric hexagonal

wrenches comes in handy when some
minor disassembly is required.

Wrenches, Open-Ended

I have an assortment of open-ended

wrenches in inch sizes as well as a metric
set. I keep two or three with the following
sizes: 1/4, 5/16, 3/8, 7/16, 1/2, and 9/16 in.,
as well as 11/16, 3/4, and 1 in., although
these latter sizes are used only rarely. I
apply two wrenches at once to prevent
counter-rotation while tightening or
loosening fittings.

19 | May 2016 | LCGC

I have one large 18-in.-long adjustable

wrench that looks like it belongs in
an automotive garage. This is used
exclusively for attaching or removing
pressure regulators on gas tanks. I also
have a smaller 6-in. long adjustable
wrench that I use occasionally if someone
else has walked off with the exact openended wrench size I need.

Chromatographers, like all craftspeople,

use a variety of tools to practice their
craft. In a pinch, tools that are somewhat
inappropriate can be used to make do,
but the rapidity and ease with which the
right tool gets the job done make it well
worth the expense of obtaining whats
needed.

References

(1) J.V. Hinshaw, LCGC North Am. 21(4), 356361 (2003).

(2) J.V. Hinshaw, LCGC North Am. 30(3), 224232 (2012).

John V. Hinshaw is a Senior Scientist at Serveron Corporation in Beaverton, Oregon, and a member of LCGCs
editorial advisory board.

How to cite this article: J.V. Hinshaw, LCGC North

America 30(7), 570577 (2012).

Pragmatic Rules
for GC Column
Selection
By Tony Taylor

GC Columns and
Consumables Primer
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view PDF

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21 | May 2016 | LCGC

In terms of selecting an appropriate stationary

phase for gas chromatography (GC), there
are four primary analyte or stationary-phase
interactions that need to be considered.
Dispersive interactions (<<1 kJ/mol) are
lower energy (van der Waals) forces between
nonpolar moieties of the analyte molecule, that
is, C-H bonds and so on. These will be in play
when using any silica-based stationary phase
because the majority of the polymeric backbone
(polydimethylsiloxane [PDMS]) of a silica-based
stationary phase is nonpolar in nature.
Dipoledipole and dipoleinduced dipole
interactions (3 and 1 kJ/mol, respectively) are
in play whenever unsaturated, aromatic, or
more-polar functional groups (that us, C-Cl
or C-N bonds) are present in the stationary
phase or analyte molecule. Stationary phases
containing phenyl, cyano, or trifluoro functional
groups are more polar than PDMS, and the
more of these functional groups there are, the
greater their influence is on the separation. The
increase in retention of aromatic compounds
and the relative decrease in retention of aliphatic
analytes when moving from a 5% phenylmethyl
PDMS phase to a 50% phenylmethyl PDMS
phase exemplifies this.

Joe Zugcic, Zugcic Photography

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Marine Pollution
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Troubleshooting

Hydrogen bonding interactions (19 kJ/

mol) are the strongest intermolecular
forces in capillary GC and occur whenever
the stationary phase contains cyano,
trifluoro, or (especially) hydroxyl functional
groups. This type of force is in play when
analyzing alcohols using a polyethylene
glycol or wax type phase.
Pragmatic phase-selection rules can be
summarized as follows:
Use the principles of like dissolves
like wherever possible and match the
polarity of the analyte to the polarity of
the stationary phase.
Remember that really there are only
five chemistries we need to consider
(see Figure 1). To increase retention or

100%
Dimethylpolysiloxane

Trifluoropropyl
dimethylpolysiloxane

alter selectivity based on a particular

interaction, increase the amount of
the functional group within the phase
(that is, move from a 14% to a 35%
cyanopropyl phase).
Use the least-polar phase possible
because more-polar phases bleed
more (its inherent in the chemistry).
A 5% phenyl column can be used to
screen unknown samples analyte
retention and selectivity can then be
assessed and a more appropriate
phase chosen if necessary.
A 5% phenyl, 50% phenyl, 14%
cyanopropyl, and a wax (polyethylene
glycol [PEG]) column cover the
widest range of possible interactions

Diphenyl
dimethylpolysiloxane

Cyanopropylphenyl
dimethylpolysiloxane

Polyethylene glycol (PEG) or

wax

Figure 1: The five most common GC column stationary-phase chemistries.

22 | May 2016 | LCGC

GC Column
Selection

Marine Pollution
Analysis

Troubleshooting

(stationary-phase polarities) in the

fewest number of columns.
How does one select the physical
column dimensions of length (L), internal
diameter, and film thickness (df)?
Column length affects the separation
efficiency and therefore the resolution.
Doubling column length doubles efficiency
(number of theoretical plates [N]), doubles
analysis time in isothermal separations
(1.51.75 increase if using gradient
temperature programming), doubles
column cost, and increases resolution by
a factor of 1.4. Increasing column length
is the worst way to improve the resolution
of a separation; however, when you have a
sample with many components (hundreds),
you sometimes need a long column. Select
column length according to the number of
species that need to be separated in the
sample. For two components, use a 10-m
column, and for hundreds of components,
use a 60-m or 120-m column.
Column internal diameter affects
retention and efficiency. Halve the column
internal diameter, double the efficiency,
and increase resolution by a factor of 1.4.
This will double retention time only for
isothermal separations and only if the film
thickness in not altered. The phase ratio
() is equal to the column radius (mm)
divided by 2 the film thickness (m).
Keep the phase ratio constant between
columns and the retention time will be
approximately constant. Use < 100

23 | May 2016 | LCGC

GC Column
Selection

for highly volatile analytes and > 400

for high-molecular-weight analytes or
for trace analysis. Use smaller internal
diameter columns when the separation
is dependent on the stationary phase
selectivity (analytes chemically very
similar) or when multiple components
need to be separated in shorter
timeframes. Note that the column capacity
will decrease as the column internal
diameter is reduced.
Film thickness effects retention of
analyte species, interaction with the silica
tubing, phase bleed, and column capacity.
Doubling the film thickness doubles
retention time for isothermal analysis and
increases retention by a factor of around
1.5 for temperature-programmed analysis.
Doubling film thickness increases elution
temperature by around 20 C. Use thin
films (0.10.25 m) for trace analysis or
when analytes are relatively involatile.
Use thicker films (15 m) when dealing
with volatile analytes, analytes at high
concentration, or when peak shape is
poor. Note that increasing film thickness
may compromise resolution for later-eluted
analytes (retention factor > 5) and that
phase bleed and column capacity increase
with increasing film thickness.
How to cite this article: T. Taylor, Pragramatic Rules
for GC Column Selection, LCGC North America 33(2),
150 (2015).

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